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Infection and Immunity, August 2003, p. 4313-4319, Vol. 71, No. 8
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.8.4313-4319.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

VH3 Gene Usage in Neutralizing Human Antibodies Specific for the Entamoeba histolytica Gal/GalNAc Lectin Heavy Subunit

Departments of Infectious Diseases,¹ Molecular Life Sciences,⁴ Laboratory for Molecular Science Research, Tokai University School of Medicine, Isehara, Kanagawa 259-1193,² Department of Tropical Medicine and Parasitology, School of Medicine, Keio University, Tokyo 160-8582, Japan,³ Departments of Medicine, Microbiology, and Pathology, University of Virginia, Charlottesville, Virginia 22908-1340⁵

Received 13 December 2002/ Returned for modification 13 March 2003/ Accepted 29 April 2003

A combinatorial human immunoglobulin gene library was constructed from peripheral lymphocytes of an asymptomatic Entamoeba histolytica cyst passer and screened for the production of Fab antibody to the parasite. One of the Fab clones, CP33, recognized the 260-kDa galactose- and N-acetyl-D-galactosamine (Gal/GalNAc)-specific lectin of E. histolytica. By shuffling the heavy and light chains of CP33 with the heavy and light chains of two libraries derived from the cyst passer and a liver abscess patient, 18 additional clones were obtained. Sequence analysis of the heavy-chain genes, including CP33-H, revealed that all the nearest V-segment germ lines belonged to the VH3 family (VH3-21, VH3-30, VH3-48, and VH3-53), but the levels of homology were only 85 to 95%. The closest D-segment germ line was D2-2 or D6-6, and for the J-segment the closest germ line was JH4b or JH6b. On the other hand, all the light-chain genes, including CP33-L, belonged to the V1 family, in which the closest V germ line gene was 02/012 or L5, with the J1, J2, J4, or J5 segment. CP33 and three other Fabs obtained by light-chain shuffling were purified and analyzed further. All of these Fabs recognized the cysteine-rich domain of the 170-kDa heavy subunit of the Gal/GalNAc lectin. Preincubation of E. histolytica trophozoites with these Fabs significantly inhibited amebic adherence to Chinese hamster ovary cells and also inhibited erythrophagocytosis. The ability of the neutralizing antibodies to block erythrophagocytosis for the first time implicates the lectin in phagocytosis and VH3 antibodies in defense against parasitic infections. These results demonstrate the utility of a combinatorial human immunoglobulin gene library for identifying and characterizing neutralizing antibodies from humans with amebiasis.

Editor: J. M. Mansfield

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